Acridine BenzimidazoliumDerivatives Induced ProtectiveMicroglia Polarization and <i>In Silico</i> TDP-43 InteractionPotentialImplications for Amyotrophic Lateral Sclerosis

Abnormal protein aggregation and associated neuronal-glial cell cytotoxicity lead to a plethora of neurodegenerative disorders. Most of the earlier investigations on understanding neurodegenerative disease progression and cure focused on neuronal damage and restoration potential. With increased evidence on the role of glial cells like microglia and astrocytes in mediating these disorders, more studies are dedicated to understanding the role of inflammatory responses mediated by glial cells and how they lead to neuroinflammation. Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder caused by TDP-43 aggregation that affects motor neurons. Pro-inflammatory microglia are considered to aggravate the disorder condition. In the current study, a previously reported molecule with TDP-43 inhibition, 3,3′-(acridine-4,5-diylbis(methylene))bis(1-(carboxymethyl)imidazol-3-ium) dibromide salt (<b>AIM4</b>), is analyzed for its microglia polarization properties along with two other derivatives, 3,3′-(acridine-4,5-diylbis(methylene))bis(1-(2-ethoxy-2-oxoethyl)benzimidazol-3-ium) dibromide salt (<b>ABE</b>) and 3,3′-(acridine-4,5-diylbis(methylene))bis(1-(carboxymethyl)benzoimidazol-3-ium) dibromide salt (<b>ABA</b>). The 3,3′-(acridine-4,5-diylbis(methylene))bis(1-(2-ethoxy-2-oxoethyl)benzimidazol-3-ium) dibromide salt (<b>ABE</b>) and 3,3′-(acridine-4,5-diylbis(methylene))bis(1-(carboxymethyl) benzimidazol-3-ium) dibromide salt (<b>ABA</b>) display the increased ability to maintain microglial cells to anti-inflammatory state and TDP-43 binding as compared to 3,3′-(acridine-4,5-diylbis(methylene)) bis(carboxymethyl)imidazolium dibromide salt (<b>AIM4</b>). This was confirmed from total nitrite levels, mitochondria membrane potential analysis, and molecular docking studies. The selected pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) displayed decreased levels, and anti-inflammatory cytokines IL-4 and IL-10 displayed increased levels, however not very significantly, upon treatment with all acridine derivatives. The compounds were investigated on lipopolysaccharides (LPS)-triggered mouse microglial cells and Danio rerio embryos displaying no significant cytotoxicity and physiological changes (cardiac rhythm), respectively. In molecular docking studies, alanine at 315 mutated to glutamate of TDP-43 directly interacts with <b>AIM4</b>. However, π–σ interactions of the aromatic backbone of acridine in <b>ABE</b> and <b>ABA</b> with 313 phenylalanine of TDP-43 along with hydrogen bonds formed between 309, 310 glycine amino acids and imidazolium bromide side chains rendered a stronger binding of these acridine derivatives with the protein potentially inhibiting fibrillation. <b>Conclusion: ABA</b>, <b>ABE</b>, and <b>AIM4</b> maintain microglia in an anti-inflammatory state. However, more studies are required to understand its interaction with TDP-43 and the mechanism of its anti-inflammatory nature.